Effect of Z-IETD-Fmk (Caspase Inhibitor) supplementation on apoptosis like changes developed in buffalo bull sperm during cryopreservation

Present study was conducted to evaluate the anti-apoptotic effect of caspase inhibitor (ZIETD-FMK) supplementation in buffalo bull semen. The Z-IETD-FMK was supplemented with Tris egg yolk extender @ 2, 4, 6, 10 and 20 µM. The pre-freeze and post thaw samples were evaluated in terms of % individual motility, % viability, % HOS reactive sperms, status of mitochondrial membrane potential and status of sperm membrane phosphatidylserine. There was no significant effect (P>0.05) of Z-IETD-FMK treatment on sperm motility (%), sperm viability (%) and percent sperm with active mitochondria at pre-freeze and post-thaw stages. However, there was improvement in terms of % HOS reactive sperms and % sperms with low PLA activity in higher supplementation doses (20 µM) of Z-IETD-FMK in post freeze semen samples as compared to control. Thus Z-IETDFMK shows anti-apoptotic effect in higher doses.

Original Research Article https://doi.org/10.20546/ijcmas.2019.802.059

Effect of Z-IETD-Fmk (Caspase Inhibitor) Supplementation on Apoptosis Like Changes Developed in Buffalo Bull Sperm during Cryopreservation

Jasmer Dalal 1* , AjeetKumar 2 , M Honparkhe 1 and P.S Brar 1

1

Department of Veterinary Gynaecology and Obstetrics, Lala Lajpat University of Veterinary

and Animal University, Hisar, 125004, India

2

Department of Veterinary Gynaecology and Obstetrics, Guru Angad Dev Veterinary and

Animal Sciences University, Ludhiana 141 004, Punjab, India

*Corresponding author

A B S T R A C T

Introduction

Artificial insemination with cryopreserved

semen is a widely used technique in buffalo

(Singh and Balhara, 2016) However, the

fertility of cryopreserved semen remains poor

(33%) as compared to fresh semen (Chohan et

al., 1992) One of the reasons for poor fertility

of cryopreserved semen is freezing induced

apoptosis like changes inflicted in

spermatozoa indicated by externalization of

phosphatidylserine (PS) due to higher

phospholipase activity (PLA) (Glander et al.,

2002) The improvement in post thaw semen quality could be done by minimizing apoptosis like changes developed during cryopreservation Apoptosis-like changes has been identified by the presence of caspase 9

and caspase 3 in bovine semen (Anzar et al.,

2002), increased membrane permeability and decreased mitochondrial membrane potential

in equine semen (Ferrusola et al., 2008) Martin et al., (2004) found that, after

cryopreservation, majority of living sperm cells showed low mitochondrial potential The caspases activate DNase and are responsible

International Journal of Current Microbiology and Applied Sciences

ISSN: 2319-7706 Volume 8 Number 02 (2019)

Journal homepage: http://www.ijcmas.com

Present study was conducted to evaluate the anti-apoptotic effect of caspase inhibitor (Z-IETD-FMK) supplementation in buffalo bull semen The Z-IETD-FMK was supplemented with Tris egg yolk extender @ 2, 4, 6, 10 and 20 µM The pre-freeze and post thaw samples were evaluated in terms of % individual motility, % viability, % HOS reactive sperms, status of mitochondrial membrane potential and status of sperm membrane phosphatidylserine There was no significant effect (P>0.05) of Z-IETD-FMK treatment

on sperm motility (%), sperm viability (%) and percent sperm with active mitochondria at pre-freeze and post-thaw stages However, there was improvement in terms of % HOS reactive sperms and % sperms with low PLA activity in higher supplementation doses (20 µM) of FMK in post freeze semen samples as compared to control Thus Z-IETD-FMK shows anti-apoptotic effect in higher doses

K e y w o r d s

Apoptosis, Buffalo

bull, Caspase

inhibitor,

Cryopreservation,

Z-IETD-FMK

Accepted:

07 January 2019

Available Online:

10 February 2019

Article Info

for DNA fragmentation (Enari et al., 1998)

and in return, DNA damage can also initiate

apoptosis (Danial and Korsmeyer, 2004)

Apoptotic sperm with fragmented DNA and

damaged membrane results in poor fertility

rates (Erickson et al., 2015) Caspases are

synthesized as inactive proenzyme

(pro-caspases) which are activated by cleavage

during the cascade of ordered events of

apoptosis (Cohen, 1997) The existence of

caspase-dependent apoptotic-like mechanisms

associated with mitochondrial functionality in

sperm, possibly similar to those found in

somatic cells (Boise and Thompson, 1997;

Ricci et al., 2003, Ricci et al., 2004: Lakhani

et al., 2006)

The Z-IETD-FMK inhibits caspase 8 (Alicia

et al., 2006) The caspase-8 has been detected

in refrigerated ram sperm samples (Mendoza

et al., 2013) Extrinsic and intrinsic are two

main pathways of apoptosis Former is

initiated by binding of extracellular death

ligand like ExoS ligand (FasL) to cellular

death receptor like Fas (Ashkenazi and Dixit

1998), later is mediated by mitochondrial

alterations Caspase-8 is a key connecting link

between two propagation pathways of

apoptosis, especially when stimulated by

external cytokines (Lee et al., 1999) So,

supplementation of caspase 8 inhibitor,

Z-IETD -FMK could be of use in minimizing

apoptosis like changes (Alicia et al., 2006)

Materials and Methods

Ethical approval

As the present did not involve handling of

live animals and no invasive technique was

used so, approval from the institutional

animal ethics committee was not required and

semen was being collected and frozen as a

routine procedure under progeny testing

program

Selection of buffalo bulls

Three breeding buffalo bull around 4 years of age maintained at bull farm, GADVASU, Punjab, India (Latitude/Longitude, 30.55°N, 75.54° E) was included in the present study These bulls were under progeny testing program and were being used for semen collection by artificial vagina method Bulls were maintained under loose housing system (covered area - 12 x 10 ft and uncovered area

- 25 x 10 ft) and standard feeding schedule along with adlib green fodder

Experimental design

Five ejaculates from each buffalo bulls were used in this study Each ejaculate was extended with Tris egg yolk extender Each ejaculate was supplemented with ZIETD -FMK in five concentrations (@ 2, 4, 6, 10 and

20 μM) Each ejaculate extended in Tris egg yolk extender and from these 6 aliquots were taken Out of these 6 aliquots, 5 were used for supplementation of Z- IETD-FMK and one was kept as control i.e without supplementation Caspase inhibitor was dissolved in dimethyl sulphoxide (DMSO) to achieve desire concentration Semen samples were frozen using traditional vapour freezing method The quality of pre-freeze and post thaw semen in terms of % individual motility,

% viability, % HOST reactive sperms, % active mitochondria and % sperm with low PLA activity (non-apoptotic sperms) were evaluated

The % individual motility was assessed manually under 20 x objective of phase contrast microscope (Nikon Eclipse E 200) The live sperm count was determined through Eosin-Nigrosin staining technique as per

standard procedure (Blom et al., 1977) The

HOS test was performed as per standard procedure to assess the functional integrity of

sperm membrane (Jeyendran et al., 1984; Dalal et al., 2016)

Evaluation of mitochondrial membrane

supplemented pre-freeze and post thaw

semen

Mitochondrial membrane potential was

assessed by using fluorescent dye

Tetra-methylrhodamine, methyl ester (TMRM, Life

Technologies; Cat#T-668) as described

previously (Dalal et al., 2016; Dalal et al.,

2018; Dalal et al., 2018a; Dalal et al., 2018b)

Briefly, semen samples (pre-freeze and post

thaw; 250 μL) were given 2 washings with

PBS by centrifuging at 1000 RPM for 5 min

at 370C Then, 5 μL of 50 μM TMRM

solution in DMSO was added to each sample

and incubated at 370C for 90 min After

incubation, washing was done with PBS at

1000 RPM for 5 min at 370C to remove all the

unbound dye The sperm pellet was mixed

well with 500 μl of PBS On a microslide, 10

μL of washed sample and 8 μL of ProLong

Gold Antifade Mountant with DAPI (Life

Technologies, Cat# P36941) was taken and

covered with coverslip The slide was

examined under upright fluorescent

microscope (Nikon) with DAPI filter

(420-480 nm), FITC FILTER (510 - 580nm) and

TRITC filter (530-580nm) Around 100

sperms were observed for high or low

fluorescence in mid piece region as an

indicator of mitochondrial membrane

potential

Evaluation of sperm phospholipase activity

in caspase inhibitors supplemented

pre-freeze and post thaw semen

Sperm phospholipid membrane was studied

using BODIPY C11 fluorescent dye

(4,4-

difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (BODIPY C11

FL, Life technologies, Cat# D 3862) as

described previously (Dalal et al., 2016; Dalal

et al., 2018; Dalal et al., 2018a; Dalal et al.,

2018b) Briefly, semen samples (pre-freeze

and post thaw; 250 μL) were given 2 washings with PBS by centrifuging at 1000 RPM for 5 min at 370C Then, 30 μL of 20

μM BODIPY solution in DMSO was added to each semen sample and incubated for 45 min

at 37 0C After incubation, washing was done with 1ml of PBS at 1000 RPM for 5 min at

370C to remove all the unbound dye The pellet was mixed well with 500 μL of PBS

On a micro slide, 10μL of sample and 8 μL of ProLong Gold Antifade Mountant with DAPI (Life Technologies, Cat# P36941) was taken and covered with cover slip Glass slides were examined under upright fluorescent microscope (Nikon) with DAPI filter

(420-480 nm), FITC filter (510 - 580nm) and TRITC filter (530-580nm) Around 100 sperms in different fields were observed and normal sperm without fluorescence were calculated out of hundred and taken as % sperm with low PLA (phospholipase A1 and A2) activity

The data was analyzed for one-way analysis

of variance (ANOVA) and Games Howell Post hoc test using IBM SPSS Version 20

Results and Discussion

In our study, Tris extender was supplemented with Z-IETD-FMK (caspase inhibitor) in the final concentration at 2, 4, 6, 10, and 10 μM and evaluated the pre-freeze and post-thaw semen samples in terms of percent individual motility, viability, HOST reactive sperms, mitochondrial membrane activity, and sperm PLA activity status Data obtained was analyzed and presented in Table 1

Sperm motility

There was no significant (P > 0.05) difference

in terms of % motility in pre-freeze Z-IETD-FMK treated and control groups as shown in Table 1 Similarly, post thaw % motility in IETD-FMK treated and control groups were

similar (P > 0.05) as shown in Table 1 The

mechanisms of inducing apoptosis by

different caspases are more complex and

many factors are involved (Sule et al., 2013)

In our study, Z-IETD -FMK did not affect %

sperm motility Chen et al., (2006) reported

the inverse relationship between sperm

motility and apoptosis in human spermatozoa

Previously, also it has been reported that

sperm motility was similar when treated with

different concentration of Z-DEVD-FMK

(Dalal et al., 2018a) and Z-LEHD -FMK

(Dalal et al., 2018b)

Sperm viability

There was no significant (P > 0.05) difference

in % sperm viability in pre-freeze IETD-FMK

treated and control groups (Table 1)

Similarly, percent viable sperms in post thaw

samples were similar (P ˃ 0.05) between

Z-IETD -FMK treated and control groups

However, in previous studies sperm viability

was improved (P<0.05) in lower doses of

DEVD-FMK (Dalal et al., 2018a) and

Z-LEHD -FMK (Dalal et al., 2018b)

Hypo osmotic swelling test (HOST)

In pre-freeze samples, there were no

significant (P < 0.05) difference between

control and Z-IETD -FMK supplemented

doses (2, 4, 6, 10 and 20 µM) in terms of %

Host reactive sperms The % Host reactive

sperms in post thaw samples were

significantly (P<0.05) higher @ 20 µM

(71.19±5.89) than control (59.33±5.22) The

% Host reactive sperms were similar in post

thaw samples significantly among 2

(62.64±5.09), 4 (65.12±6.83), 6 (63.18±7.44)

and 10µM (65.29±5.61) and did not differ

significantly from control and 20 µM treated

group In previous study, Dalal et al., (2018b)

demonstrated that 2µM of Z-DEVD -FMK

protect the functional integrity of sperm

plasma membrane In another study with

other caspase inhibitor, Dalal et al., (2018a)

reported that Z-LEHD-FMK in lower dose (2 and 4µM) protects the functional integrity of buffalo sperm

Mitochondrial status

In pre-freeze samples, there were no significant (P > 0.05) differences between control (without Z-IETD -FMK) and supplemented (2, 4, 6, 10 and 20 µM doses of Z-IETD -FMK) in terms of % active mitochondria

In post thaw samples, the % active mitochondria were also similar (P> 0.05) in Z-IETD -FMK supplemented and control groups Hence, supplementation of Z-IETD-FMK did not affect mitochondrial membrane potential of spermatozoa following cryopreservation of semen However, in

previous studies Z-DEVD-FMK (Dalal et al., 2018a) and Z-LEHD -FMK (Dalal et al.,

2018b) improved the mitochondrial potential

PLA activity

In pre-freeze semen samples, there were no significant (P < 0.05) difference between control and Z-IETD-FMK supplemented doses (2, 4, 6, 10 and 20 µM) in terms of % sperms with low PLA activity

The % sperms with low PLA activity in post thaw samples were significantly (P<0.05) higher @ 20 µM (76.56±5.23) of Z-IETD-FMK than control (61.56±5.98) The % sperms with low PLA activity were similar in post thaw samples significantly among 2 (63.44±6.37), 4 (65.11±5.88), 6 (69.71±8.55) and 10µM (73.19±6.28) of Z-IETD-FMK and did not differ significantly from control and

20 µM treated group

Our study indicated that Z-IETD-FMK supplementation has protective effect against

apoptosis like changes in spermatozoa during

cryopreservation especially in higher doses

(20 µM) as reported previously with

DEVD-FMK(Dalal et al., 2018a) and

Z-LEHD-FMK (Dalal et al., 2018b) but in these

study effect was also observed in lower doses indicating that Z-DEVD-FMK and Z-LEHD-FMK inhibitors are more potent than Z-IETD-FMK in protecting the sperm from cryopreserved induced apoptosis

Table.1 Effects of supplementation of Z-IETD-FMK at various concentrations at pre-freeze and

post thaw stage

Motility 91.12±

6.23a

85.23±

5.48a

85.51±

6.24a

90.76±

7.11a

86.34±

5.89a

90.34±

6.75a

50.55±

5.38a

55.57±

7.79a

50.43±

8.34a

52.46±

5.45a

45.23±

6.88a

45.29± 5.78a

%

Viability

90.56±

4.33a

95.17±

5.88a

89.81±

7.73a

88.62±

6.89a

80.56±

7.49a

85.45±

5.85a

60.22±

6.47a

65.48±

6.31a

55.87±

7.39a

58.11±

5.29a

60.81±

6.39a

67.73± 4.59a

% HOS

reactive

sperm

82.56±

3.45a

83.46±

5.76a

81.37±

7.23a

85.68±

5.91a

88.14±

4.38a

85.18±

3.84a

59.33±

5.22a

62.64±

5.09 a

65.12±

6.83 ab

63.18±

7.44 ab

65.29±

5.61 ab

71.19± 5.89 b

% Active

mitochon

dria

81.78±

7.99a

83.45±

3.49a

75.55±

4.64a

78.42±

5.32a

75.12±

3.56a

78.68±

4.88a

55.52±

5.87a

60.71±

4.99a

52.53±

6.85a

57.48±

4.68a

50.83±

5.83a

57.44± 6.24a

%

sperms

with low

PLA

activity

78.34±

3.48a

75.31±

5.77a

81.45±

4.37a

85.28±

6.27a

86.45±

5.37a

88.11±

4.77a

61.56±

5.98 a

63.44±

6.37ab

65.11±

5.88 ab

69.71±

8.55 ab

73.19±

6.28 ab

76.56± 5.23b

To the best of our knowledge, this is the first

report on use of Z-IETD-FMKto minimize

apoptosis like changes in buffalo sperm

induced during cryopreservation

Cryopreservation induce increase in caspase

activation in human sperm positive for active

Caspase-3 (32.6%) followed by active

Caspase-8 sperm (30.5%), active Caspase-9

sperm (22.2%) and active Caspase-11 sperm

(15.5%) underlining the central role of the

effector caspase-3 (Paasch et al., 2004) The

increase in caspase activation is dependent on

the sperm preparation and cryopreservation

protocol (Grunewald et al., 2005) and each

species has its own optimum freezing rates

(Dalal et al., 2018) Cryopreservation has

been reported to activate caspase-3 and -9 in

humans (Paasch et al., 2004; Bejarano et al., 2008) and in boar sperms (van Gurp et al.,2003) Caspase activation following the

cryopreservation and thawing process is also

reported in cattle (Martin et al., 2004;Martin

et al.,2007) and equine spermatozoa (Brum et al., 2008; Ferrusola et al., 2008)

The Z-IETD-FMK is a powerful cell permeable selective inhibitor of caspase 8

(Alicia et al., 2006) Caspase 8 is involved in

the membranous pathway of apoptosis After stimulation by external death signals,

caspase-8 can directly either activate executioner

caspases or convert BID (BH3 interacting

domain) into tBid (truncated BID), which

translocate from cytosol to mitochondria

where it enhances permeability and release of

cytochrome c (Lee et al., 1999) Thus,

Caspases 8 act through membranous pathway

of apoptosis (Slee et al., 1999) and

membranous pathway is less commonly

operated in bovine sperm (Zou et al., 1999)

However, Z-IETD-FMK partially inhibits the

caspase 3 as reported treatment of retinal cells

with 20µM of the selective inhibitor of

caspase-8, Z-IETD-FMK, resulted in

decreased caspase-3like activity only in

retinal cells (Gülgün and Martin, 1999) In

present study also, Z-IETD-FMK showed

antiapoptotic effect but in higher

concentration (20µM) suggesting that in

lower concentration it partially inhibit caspase

3 However higher concentration of

Z-IETD-FMK might cause more potent inhibitory

effect on caspase 3

Our study indicates that higher concentration

(20 µM) of Z-IETD –FMK protect the sperm

membrane integrity and prevented

externalization of phosphatidylserinesperm

with low PLA activity of sperm based on the

fact that spermatozoa with deteriorated

phosphatidylserine are characterized by an

increased lyso-phosphatidylcholine content

that is likely generated by phospholipases

(Glander et al., 2002) Furthermore,

additional depth studies will be required to

assess the other properties of caspase

inhibitors to revealed exact mechanisms of

actions It has been reported that the addition

of caspase inhibitors to the cryopreservation

medium failed to improve the acrosome and

plasma membrane integrity of frozen-thawed

of ram (Marti et al., 2008), dog(Peter and

Linde-Forsberg, 2003) and stallion sperms

(Peter et al., 2005) These differences from

our study may be due to the species variation

or differences in doses of supplementation

Peter et al., (2005) also suggested that a

higher or lower level of caspase doses with different timings of treatment may produce the desired effects

In conclusion, the Z-IETD -FMK improves spermplasma membrane integrity in higher concentrations together with maintenance of low PLA activity in post thaw sperm It implies that apoptosis like changes developed during cryopreservation and Z-IETD -FMK helps to counteract these apoptosis-like changes in sperms

Conflict of interest statement

The authors declare no conflict of interests (financial or nonfinancial) with any organization or entity

Acknowledgments

This work was supported by the Department

of Biotechnology (DBT), Government of India; vide BT/PR3596/AAQ/01/483/2011 under the project “Improvement in fertilizability of cryopreserved buffalo bull semen by minimizing cryo-capacitation and apoptosis-like changes.”

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How to cite this article:

Jasmer Dalal, AjeetKumar, M Honparkhe and Brar, P.S 2019 Effect of Z-IETD-Fmk (Caspase Inhibitor) Supplementation on Apoptosis Like Changes Developed in Buffalo Bull

Sperm during Cryopreservation Int.J.Curr.Microbiol.App.Sci 8(02): 516-524

doi: https://doi.org/10.20546/ijcmas.2019.802.059